Tissue regeneration, remodeling, and homeostasis are fundamental biological processes shared by all animals, and when their regulation is compromised, human diseases such as cancers and degenerative disorders often result. However, our molecular understanding of these processes has been hampered in great part by the extremely limited regenerative capabilities of established invertebrate model systems. The experiments put forth in this proposal fulfill the mission of the NIH and are aimed at achieving our long-term objective to understand how undifferentiated somatic stem cells communicate with their surrounding cells in adult animals. These experiments take advantage of planarians, masters of regeneration. One hundred planarian genes from seven conserved cell signaling pathways will be analyzed for roles in tissue regeneration, remodeling, and homeostasis. While many of these genes are mutated in different forms of human cancer, their precise functions in healthy adult animals are unknown. This is because null mutations in the implicated genes cause embryonic lethality in other organisms, making it difficult to study these pathways in adults. In contrast, planarians provide an in vivo platform for the rapid, systematic analysis of gene function in adult animals. In Aim 1, planarian orthologs including ligands, receptors, effectors, transcription factors, cofactors, targets, and antagonists from seven highly conserved and developmentally relevant signaling pathways will be identified and cloned. Expression of these genes will be analyzed in intact and regenerating animals by whole-mount in situ analysis and in FACS purified cell populations. In Aim 2, these genes will be silenced in planarians by RNA interference (RNAi) and the animals will be screened for defects in tissue regeneration, remodeling, and homeostasis. In Aim 3, expression patterns and phenotypic data derived from Aims 1 and 2 will be used to inform the simultaneous silencing of multiple genes. Aim 3 will potentially identify synergism and interdependence between different signaling pathways that may be specific to regenerative processes and may be involved in cancer formation and/or progression. The experiments described in this proposal are aimed at understanding how stem cells in adult animals form structures of appropriate form and function during 1) tissue regeneration, 2) tissue remodeling after injury, and 3) normal cell turnover. The investigation focuses on these processes in planarians, animals with the amazing power to regenerate any missing structure after injury. Because highly conserved signaling pathways will be examined, this work has the potential to impact the fields of cancer, regenerative medicine, and stem cell biology among others.